To elucidate the characteristics of glycoproteins in the mesangial matrix in glomeruli from patients with diabetic nephropathy (DN) in non-insulin-dependent diabetes mellitus (NIDDM), we examined periodic acid-Skiff (PAS) and immunofiuorescence staining of types III (III-C), IV (IV-C), V (V-C) and VI (VI-C) collagen, fibronectin (FN), fibrinogen (FBG) and laminin (LN) in the glomeruli of 16 patients with DN and 12 patients with diffuse proliferative glomerulonephritis (DPGN). The percentages of positively staining areas in the glomeruli were calculated using an automatic image analyzer. The percentages of areas positive for PAS, III-C, IV-C, V-C, VI-C or FN in DN were significantly greater than those in DPGN. Moreover, the percentages of PAS-positive areas were significantly correlated with not only IV-C-positive areas, but also FN-positive areas in patients with DN. The percentages of PAS-positive areas were also significantly correlated with the levels of serum creatinine and the degree of proteinuria in these patients. It was concluded that mesangial expansion in this disease might be associated mainly with an increase in IV-C and FN. These changes appear to affect the deterioration of renal function and the appearance of proteinuria, and vice versa.

View full abstract

We evaluated the effects of a novel calcium antagonist, manidipine, on albuminuria in rats with passive Heymann nephritis (PHN). Treatment with 0.05% manidipine significantly reduced urinary albumin excretion (62.1±7.5 vs. 46.9±8.5 mg/urinary ereatinine excretion mg, P<0.05) and attenuated lipid peroxidation of the renal cortices (0.97±0.08 vs. 0.84±0.11 nM MDA/mg protein, P<0.05) on day 14 in PHN. Manidipine affected neither the light microscopic, immunofluorescent nor electron microscopic findings. These results indicate that manidipine reduced proteinuria in rats with PHN, and that its antiproteinuric effect was associated with the reduction of lipid peroxidation.

View full abstract

We studied DNA synthesis in rats with nephrotoxic serum nephritis (NTSN), a model of a glomerular disease, using in vivo labelling with 5-bromo-2'-deoxyuridine (BrdUrd). NTSN was induced by intravenous injection of subnephritogenic doses of rabbit anti-rat GBM antiserum into male Sprague-Dawley rats. Each rat received a single injection of the DNA precursor, 3H-thymidine analog (BrdUrd), ten min before the tissues were removed, For Immunocytochemical detection of DNA synthesis, semithin sections were prepared at various intervals (4 h up to 84 days) after pulse labelling . Using a monoclonal anti-BrdUrd antibody, BrdUrd-incorporated DNA-synthesizing cells were noted in the proliferative zone of the gastric mucosa at all times. In NTSN, BrdUrd incorporated DNA-synthesizing cells were detected in the glomeruli from 4 h through 28 days after inoculation, with the peak occurring at days 2 to 4. On those days, up to half of the glomeruli showed BrdUrd-incorporated cells, with 8 cells per glomerulus as a maximum. From days 7 to 28, few glomerular cells incorporated BrdUrd, and none did so after day 28. The majority of the BrdUrd-incorporated cells were endothelial . These results suggest that active DNA synthesis by glomerular endothelial cells occurs during a short period of the heterologous phase in this model, and that the lack of mesangial cell proliferation might explain the selflimiting nature of this model. By using in vivo labelling with BrdUrd, we were also able to easily and accurately detect active DNA synthesis without consideration of the normal cell renewal . BrdUrdimmunohistochemistry may be an alternative nonradioactive, highly sensitive approach for detecting glomerular cell proliferation, even in mild NTSN.

View full abstract

The current studies were designed to identify which mononuclear leukocytes have an important role in the development of glomerular injury using rats with original-type (mild injury) and crescentictype (severe injury) anti-glomerular basement membrane (GBM) nephritis. 1) Proteinuria was persistent in crescentic-type anti-GBM nephritis compared with original-type anti-GBM nephritis. Macrophages/monocytes (ED-1), cytotoxic/suppressor T cells (CD-8), interleukin-2-receptor (CD-25)-positive cells and Ia-positive cells accumulated remarkably and persisted for longer in crescentic-type nephritic glomeruli. 2) We then performed investigations using immunosuppressants. Cyclosporin A abrogated proteinuria more effectively than azathioprine in crescentic-type nephritis. However, plasma antibody titer and glomerular rat lgG deposition were equally reduced by both azathioprine and cyclosporin A. The increase in the numbers of ED-1-, CD-8- and CD-25-positive cells in nephritic glomeruli was completely inhibited by cyclosporin A, but inhibited only slightly by azathioprine. 3) There was a correlation between the degree of proteinuria and the number of ED-1- and CD-8-positive cells. It is likely that these cells are leukocytes that lead to glomerular injury in nephritis. 4) In additional experiments using monoclonal antibodies against macrophages/monocytes and cytotoxic/suppressor T cells, urinary protein excretion and accumulation of these cells were blunted in nephritic rats treated with these antibodies. These results suggest that ED-1- and CD-8-positive cells are involved in the development of crescentic-type anti-GBM nephritis.

View full abstract

Alterations of Na⁺-dependent D-glucose transport in the proximal tubules resulting from ischemic injury were investigated. We measured the cortical oxygen (O₂) consumption at various concentrations of exogenous D-glucose 1 day after ischemic injury in a kidney in which ichemia was induced by complete unilateral renal artery occlusion for 60 minutes and in a contralateral non-ischemic (control) kidney. Both kidneys exhibited a D-glucose concentration-dependent increase in O₂ consumption. The increases in O₂ consumption in both kidneys exhibited Michaelis-Menten-type saturation kinetics. However, increased K_m and decreased V_max were observed in the ischemic kidney. Phlorizin significantly inhibited O₂ consumption in both kidneys at 5mM, but the effect was not significant at 0mM of D-glucose. In contrast, ouabain significantly inhibited O₂ consumption in both kidneys at 0 and 5 mM of D-glucose. The level of O₂ consumption inhibition by phlorizin and ouabain in the ischemic kidney was markedly less than in the control kidney. The present study demonstrated that the decreased Natdependent D-glucose transport capacity can be attributed to a reduction in both phlorizin-sensitive transport capacity for D-glucose and ouabain-sensitive Na⁺, K⁺-ATPase activity 1 day after 60 minutes of ischemic.

View full abstract

To provide evidence on renal gluconeogenesis in humans and to compare with rats and rabbits, glucose production from several substrates was determined using cortical slices of the three species. In humans, the normal parts of kidney tissue were obtained from six cases of transitional cell carcinoma of the renal pelvis and four cases of renal cell carcinoma. Renal cortical slices were incubated aerobically with or without substrates, and the glucose contents were assayed photometrically. The specificity of human kidneys was evaluated by comparison with the results obtained from rats and rabbits. The rank order of renal gluconeogenesis from various substrates was as follows: Humans: pyruvate>oxaloacetic acid>lactic acid> fructose-l, 6-diphosphate>L-glutamine. Rats: pyruvate>fructose-l, 6-diphosphate>oxaloacetic acid>lactic acid>L-glutamine. Rabbits: fructose-1.6-diphosphate> pyruvate>oxaloacetic acid>lactic acid>>L-glutamine=0. In general, the human kidney can produce glucose at the lowest rate among the three species. The substrate specificity of humans was more or less similar to that of rats. These results suggest the existence of a species difference in renal gluconeogenesis both in substrate specificities and quantitative activities.

View full abstract

Advanced glycosylation end products are believed to play a role in the increase in extracellular matrix in diabetic glomerulosclerosis via a pathway involving a mesangial cell fibronectin receptor. In the present study, I assessed cultured rat mesangial cells for the presence of mRNA for VLA5, one of the fibronectin receptors. Using these cells, I also evaluated the effects of glycated proteins on the expression of VLA5 and fibronectin. Mesangial cells of Wistar rats were cultured in RPMI 1640 containing 20% FCS. The cells were incubated for 72 hr in a medium containing 50 mg/ml of nonglycated BSA or in a medium containing 50mglml glycated BSA (AGE?EBSA). Immunostaining and Northern blot analyses showed that the cells incubated with AGE BSA exhibited a decrease in VLA5 protein and related mRNA, but showed an increase in the synthesis of fibronectin and related mRNA. On the other hand, non-glycated BSA did not induce these changes in the expression of VLA5 or fibronectin in the mesanginal cells. Northern blot analysis for VLA5 mRNA was also conducted using mesangial cells cultured in a medium with a high glucose concentration (30mM). However, the high glucose condition did not have any effect on the expression of VLA5 protein or related mRNA. These results suggest that glycated proteins may regulate fibronectin synthesis in mesangial cells by modifying the expression of fibronectin receptors, such as the inhibition of VLA5 synthesis that acts as negative feedback of fibronectin synthesis.

View full abstract

The effects of puromycin aminonucleoside (PAN) on rat glomerular epithelial cells (GECs) in primary culture were studied using a quick-freezing and deep-etching method. On the 5th day after seeding, GECs were treated with PAN for 24 hours. The three-dimensional structure of the cell organelles, microtubules, and intermediate filaments were observed, primarily in the perinuclear areas of untreated GECs. Microfilaments were observed in marginal and basal areas. The GECs treated with PAN became rounded and partially detached from the substratum. Intermediate filaments formed bundled structures. A slight decrease in the number of microfilaments and a significant widening of their networks were observed in the basal areas of the GECs treated with PAN. These results suggest that PAN may affect the cytoskeletal components of GECs, such as the intermediate filaments and microfilaments, and may induce detachment of the cells from the substratum.

View full abstract

The effect of drugs that alter the renal tubular transport of urate in the renal excretion of the oxypurines, hypoxanthine (Hx) and xanthine (Hx), was used to analyze the tubular mechanisms involved in oxypurine excretion in normal subjects and in patients with idiopathic renal hypouricemia. In healthy subjects, administration of the drugs, benzbromarone (Bb) and probenecid (Pb), brought a marked uricosuric effect, but did not alter oxypurine excretion. One of 2 hypouricemic patients exhibited no uricosuric effects with Bb and the other showed a slight uricosuric effect with the drug. Bb administration, however, produced no increase in oxypurine excretion in either patient. In healthy subjects, adminis tration of pyrazinamide (PZA) suppressed the excretion of urate and oxypurines to varying degrees: relative to the baseline values, the fraction excretion of urate was reduced by 95% or more, that of Hxby about 24%, and that of X by about 64%. In patients with hypouricemia, the effects of PZA on urate and oxypurine excretion were impaired. According to these findings, we can speculate that the oxypurines have the same renal secretory mechanism as uric acid. However, the reabsorptive mechan isms of oxypurines at the postsecretory site are likely to differ from that of uric acid.

View full abstract

A case of light chain deposition disease with multinucleated giant cell accumulation in the kidney is described. A 54-year-old man was admitted to out hospital due to moderate renal failure. Complicated with eosinophilic pneumonia two months after admission, his renal function abruptly deteriorated and hemodialysis was started. Three-day methylprednisolone pulse therapy, however, partially recovered his renal function and hemodialysis was discontinued. His renal biopsy specimen revealed kappa light chain deposition disease with nodular mesangial expansion and subendothelial electron dense deposits. The most characteristic finding in this patient was accumulation of foreign body type multinucleated giant cells around atrophic tubules, small arteries and obsolescent glomeruli, probably associated with light chain deposition. No increase in kappa light chain was detected in his serum or concentrated urine. Such disseminated giant cell reaction, other than intra-luminal infiltration in the tubules, has not been reported in the literature and might be related to the physicochemical or structural properties of the deposited protein.

View full abstract

IgA nephropathy is characterized by a predominant deposition of IgA in the glomerular mesangium. However, in many cases, deposition of IgG also occurs and the concentration of circulating IgG immune complexes is higher than that in controls. To examine further the possible role of the charge of immunoglobulins in the pathogenesis of IgA nephropathy, we used isoelectric focusing (IEF) and densitometry to investigate the charge distribution of plasma IgG and IgG immune complexes in patients with this disease. Blood samples were taken from patients and healthy adults, and plasma and samples treated with 7.0% polyethylene glycol (PEG) were used. All samples were focused with a Multiphor II flatbet electrofocusing unit apparatus on an agarosegel, then immunofixed with polyclonal goat anti-human IgG, and stained with Coomassie blue R 250. The stained gels were analyzed by densitometry.1. In plasma, the areas of PI 10-8.9 and PI 10-8.6 in IgA nephropathy were higher in the patients than in the controls. 2. In the 7.0%PEG precipitation, the area PI 8.1-6.1 was higher in the patients than in the controls. These findings suggest that a change in charge distribution of IgG and IgG immune complexes may contribute to the pathogenesis of IgA nephropathy.

View full abstract

To investigate whether the nutritional improvement achieved by recombinant human erythropoietin (rHuEPO) treatment is the result of anemia correction with rHuEPO or the direct anabolic effects of rHuEPO per se, nutritional assessment was performed in 2 studies (study I and II) on hemodialysis (HD) patients. Nutritional assessment included blood biochemistry determinations, anthropometric measurements, daily protein intake (DPI) and dialysis efficiency. In study I, 5 HD patients who had not been given rHuEPO and had a hematocrit (Hct) of ≤25%, were administered rHuEPO at the initial dose of 96.2U/kgBW. Nutritional assessment of these patients was performed before rHuEPO treatment and every 4 weeks until the 24th week after rHuEPO treatment. In study II, the same nutritional assessment as in study I except for DPI, was performed in 2 groups with the same Hct level and dialysis regimen; an EPO group (n=8) previously given rHuEPO (88.2±13.7 U/kgBW, 25.8±2.5mos) and a non-EPO group (n=8) not given rHuEPO. In study I, the mean Hct level was significantly increased 4 weeks after rHuEPO treatment (23.3±0.6 to 26.9±0.9%). However, the nutritional parameters and dialysis efficiency were nearly constant over 24 weeks, suggesting either the absence of a short-term direct anabolic effect of rHuEPO or masking of such an effect due to general condition improvement by anemia correction with rHuEPO. In study II, no significant differences in nutritional assessment were confirmed between the groups, suggesting that a long-term direct anabolic effect of rHuEPO may not exist and nutritional improvement may result from correction of anemia with rHuEPO. We propose that dialysis efficiency will not be reduced by anemia correction of at least Hct≤30% with rHuEPO treatment.

View full abstract

The present study investigated changes in peripheral blood T cell surface markers and cytokines (interleukin-2, IL-2; tumor necrosis factor-alpha, TNF-alpha; and interferon-gamma, IFN-gamma) following tonsillectomy in patients with IgA nephropathy accompanied by chronic tonsillitis. Peripheral blood CD8⁺ cells, CD45RA⁺CD4⁺ cells and CD8⁺CD11b^- cells increased significantly after tonsillectomy, compared with their preoperative values. In some cases, the preoperative serum TNF-alpha and IFN-gamma levels were higher than normal before surgery, but decreased after surgery. These results suggest that tonsillectomy suppresses a decrease in suppressor T cells in patients with IgA nephropathy and corrects abnormal cell-mediated immune responses in these patients.

View full abstract

We present the first female patient to exhibit Fabry-like myelin bodies in the glomerular epithelial cell in association with IgA nephropathy. This previously healthy 36-year-old woman presented with proteinuria and hematuria without skin lesions. Renal biopsy showed typical IgA nephropathy, with paramesangial deposits, mesangial proliferation and scattered myelin bodies. The leukocytic α-galactosidase A activity was abnormally low . She had no family history of Fabry's disease nor the characteristic features, such as skin lesion, neuralgia, or hypohidrosis. Fabry's disease is diagnosed from the renal biopsy findings and the activity of α-galactosidase A in leukocytes and/or fibroblasts. We diagnosed the present case with Fabry' disease and IgA nephropathy from these results.

View full abstract

A 78-year-old man was hospitalized because of muscular weakness and acute renal failure. He had been taking glycyrrhizin (280mg/day) for the last 7 years. Hypertension was noted in his history. Serum potassium was 1.9mEq/l with metabolic alkalosis. There was hyporeninemic hypoaldosteronism. Serum enzymes, including GOT, LDH and CPK were markedly elevated. In addition, serum myoglobin was as high as 46μg/ml with massive myoglobinuria. Oliguria occurred and blood urea nitrogen and scrum creatinine rapidly elevated from 20.9 to 87mg/dl and from 1.3 to 6.7mg/dl, respectively. Profound calcium deposition was found in the damaged skeletal muscles, including the quadriceps femoris, axillar, neck, and cardiac muscles. These results indicate that licorice-induced pseudoaldosteronism produces hypokalemic rhabdomyolysis, resulting in acute renal failure and profound deposition of calcium into the damaged skeletal and cardiac muscles.

View full abstract

A 49-year-old woman suffering from nephrotic syndrome (NS) was admitted for renal biopsy and treatment of NS. Light microscopy demonstrated that the glomerular capillary wall was markedly thickened with diffuse accumulation of periodic acid Schiff-and periodic acid methenamine-positive materials, leading to lobular accentuation of glomerular tufts. By electron microscopy, numerous collagenous fibers were observed in the mesangium and subenodothelial area. The fibers were peculiarly curved and frayed, as reported in nail-patella syndrome. These materials were thought to be type I and type III collagen as a result of immunohistochemical studies. No laboratory data or pathological findings were found to be compatible with previously described glomerulonephritis. The primary glomerular fibrosis in the present patient seemed to be a case of collagenofibrotic glomerulonephropathy.

View full abstract

We present a 23-year-old woman with a 7-year history of insulin-dependent diabetes mellitus (IDDM) who became pregnant. At the 23th week of pregnancy she exhibited the signs and symptoms (hypertension, edema, proteinuria) of both diabetic nephropathy and preeclampsia. A cesarean section was successfully performed. The proteinuria persisted for more than 3 months after delivery. Renal biopsy confirmed the diagnosis of diabetic glomerulosclerosis together with the renal findings attributable to preeclampsia. The rapid acceleration of diabetic nephropathy in this patient was attributed to preeclampsia. We therefore recommend that patients with DM be followed closely during pregnancy in an attempt to prevent the acceleration of renal damage by preeclampsia.

View full abstract